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Literature summary for 3.2.1.40 extracted from

  • Cui, Z.; Maruyama, Y.; Mikami, B.; Hashimoto, W.; Murata, K.
    Crystal structure of glycoside hydrolase family 78 alpha-L-Rhamnosidase from Bacillus sp. GL1 (2007), J. Mol. Biol., 374, 384-398.
    View publication on PubMed
Search for more literature for 3.2.1.40

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli strain HMS174-DE3, transformation with mutant plasmids, pD567N, pE572Q, pD579N, or pE841Q for mutant generation Bacillus sp. (in: Bacteria)

Crystallization (Commentary)

Crystallization (Comment) Organism
native and selenomethionine-derivaties, 1.9 A resolution with a final R-factor of 18.2%, hanging-drop method, data-collection and statistics indicated, overall structure shown, quaternary structure, catalytic domain and active cleft determined, complex with the reaction product rhamnose determined and refined at 2.1 A with a final R-factor of 19.5% Bacillus sp. (in: Bacteria)

Protein Variants

Protein Variants Comment Organism
D567N mutagenesis of catalytic amino acid residues identifed by crystallization studies, enzyme activity reduced Bacillus sp. (in: Bacteria)
D579N mutagenesis of catalytic amino acid residues identifed by crystallization studies, enzyme activity reduced Bacillus sp. (in: Bacteria)
E572Q mutagenesis of catalytic amino acid residues identifed by crystallization studies, enzyme activity reduced Bacillus sp. (in: Bacteria)
E841Q mutagenesis of catalytic amino acid residues identifed by crystallization studies, enzyme activity reduced Bacillus sp. (in: Bacteria)

Inhibitors

Inhibitors Comment Organism Structure
L-rhamnose crystallized complex with interacting site determined, for kinetic assay different concentrations ranging between 0 and 100 mM of rhamnose used Bacillus sp. (in: Bacteria)

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information 2.5 micrograms per ml of purified protein used for the wild-type enzyme and 500 micrograms per ml for mutant enzymes Bacillus sp. (in: Bacteria)
0.28
-
 p-nitrophenyl-alpha-L-rhamnopyranoside wild-type Bacillus sp. (in: Bacteria)
0.3
-
 p-nitrophenyl-alpha-L-rhamnopyranoside E572Q Bacillus sp. (in: Bacteria)
0.31
-
 p-nitrophenyl-alpha-L-rhamnopyranoside E841Q Bacillus sp. (in: Bacteria)
0.36
-
 p-nitrophenyl-alpha-L-rhamnopyranoside D567N Bacillus sp. (in: Bacteria)
0.48
-
 p-nitrophenyl-alpha-L-rhamnopyranoside D579N Bacillus sp. (in: Bacteria)

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+ calcium ions essential for crystallization, calcium binding shown in crystallized form Bacillus sp. (in: Bacteria)

Organism

Organism UniProt Comment Textmining
Bacillus sp. (in: Bacteria) Q93RE7 rhamnosidase B, RhaB
-
Bacillus sp. (in: Bacteria) GL1 Q93RE7 rhamnosidase B, RhaB
-

Purification (Commentary)

Purification (Comment) Organism
native and recombinant proteins, gel filtration, SDS-PAGE Bacillus sp. (in: Bacteria)

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
 assayed in a reaction system containing 0.4 mM p-nitrophenyl-alpha-L-rhamnopyranoside as a substrate and 50 mM potassium phosphate buffer, resultant p-nitrophenol concentration determined spectrophotometrically Bacillus sp. (in: Bacteria)

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
gellan + H2O release of rhamnose from the disaccharide, hydrolytic reaction Bacillus sp. (in: Bacteria) alpha-L-rhamnose + ? residues interacting with rhamnose that are crucial for enzyme catalysis and/or substrate binding identified by crystallization ?
gellan + H2O release of rhamnose from the disaccharide, hydrolytic reaction Bacillus sp. (in: Bacteria) GL1 alpha-L-rhamnose + ? residues interacting with rhamnose that are crucial for enzyme catalysis and/or substrate binding identified by crystallization ?
naringin + H2O bitter flavour source in fruit Bacillus sp. (in: Bacteria) 4',5,7-trihydroxyflavanone-7-beta-D-glucoside + alpha-L-rhamnose
-
 ?
naringin + H2O bitter flavour source in fruit Bacillus sp. (in: Bacteria) GL1 4',5,7-trihydroxyflavanone-7-beta-D-glucoside + alpha-L-rhamnose
-
 ?
p-nitrophenyl-alpha-L-rhamnopyranoside + H2O assay reaction system to determine activity of wild-type and mutant enzymes Bacillus sp. (in: Bacteria) p-nitrophenol + alpha-L-rhamnopyranose
-
 ?
p-nitrophenyl-alpha-L-rhamnopyranoside + H2O assay reaction system to determine activity of wild-type and mutant enzymes Bacillus sp. (in: Bacteria) GL1 p-nitrophenol + alpha-L-rhamnopyranose
-
 ?

Subunits

Subunits Comment Organism
homodimer crystallized form, 1908 amino acids of residues 3-956, 43 glycerol molecules, four calcium ions, 1755 water molecules identified in the crystallized form Bacillus sp. (in: Bacteria)

Synonyms

Synonyms Comment Organism
alpha-L-rhamnosidase
-
 Bacillus sp. (in: Bacteria)

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
 assay at Bacillus sp. (in: Bacteria)

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
 assay at Bacillus sp. (in: Bacteria)

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.8
-
 L-rhamnose crystallized complex with interacting site analyzed Bacillus sp. (in: Bacteria)